Coronaviruses are a family of enveloped RNA viruses that cause respiratory, enteric, and neurologic diseases in mammalian and avian hosts. In humans, four coronaviruses are responsible for common upper respiratory tract infections;a fifth human coronavirus is the causative agent of severe acute respiratory syndrome (SARS), which has the potential to re-emerge into the human population from animal reservoirs. To manipulate the genomes of coronaviruses, which are the largest among all the RNA viruses, our laboratory developed the earliest reverse genetic system, using the prototype coronavirus mouse hepatitis virus (MHV). This method transduces targeted site-specific mutations into the MHV genome via recombination with synthetic RNA introduced into infected cells. Coupled with a powerful host-range-based selection system, targeted RNA recombination has become a robust and versatile technique that has been used to answer fundamental questions about viral protein structure and function, host species specificity, virion assembly, and the complex mechanism of coronavirus RNA synthesis. The major object of this proposal is to study processes that are central to multiple phases of coronavirus replication. Targeted RNA recombination, other reverse genetic methodologies, and complementary biochemical and molecular biological analyses will be employed: (i) to investigate a newly discovered critical interaction between the nucleocapsid (N) protein and a component of the viral replication-transcription complex;(ii) to elucidate the crucial role of N protein in the initiation of coronavirus infection and how this role is modulated by phosphorylation of N protein;and (iii) to dissect the network of structural protein interactions among N protein, the membrane (M) protein, and the small envelope (E) protein that are essential to virion assembly and the packaging of genomic RNA.